Fuel ethanol is a suitable alternative to fossil fuels. Ethanol may be produced from plant biomass, which is an economical and renewable resource that is available in large amounts. Examples of biomass include agricultural feedstocks, paper wastes, wood chips and so on. The sources of biomass vary from region to region based on the abundance of natural or agricultural biomass that is available in a particular region. For example, while sugar cane is the primary source of biomass used to produce ethanol in Brazil, corn-derived biomass, corn starch is a large source of biomass to produce ethanol in the United States. Other agricultural feedstocks include, by way of example: straw; grasses such as switchgrass; grains; and any other lignocellulosic or starch-bearing material.
A typical biomass substrate contains from 35-45% cellulose, 25-40% hemicellulose, and 15-30% lignin, although sources may be found that deviate from these general ranges. As is known in the art, cellulose is a polymer of glucose subunits, and hemicellulose contains mostly xylose. Arabinose is also a significant fermentable substrate that is found in biomass, such as corn fiber and many herbaceous crops in varying amounts. Other researchers have investigated the utilization of arabinose and hemicellulose, as reported by Hespell, R. B. 1998. Extraction and characterization of hemicellulose from the corn fiber produced by corn wet-milling processes. J. Agric. Food Chem. 46:2615-2619, and McMillan, J. D., and B. L. Boynton. 1994. Arabinose utilization by xylose-fermenting yeasts and fungi. Appl. Biochem. Biotechnol. 45-46:569-584. The two most abundant types of pentose that exist naturally are D-xylose and L-arabinose.
It is problematic that most of the naturally available ethanol-producing microorganisms are only capable of utilizing hexose sugar, such as glucose. This is confirmed by a review of the art, such as is reported by Barnett, J. A. 1976. The utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem. 32:125-234. Many types of yeast, especially Saccharomyces cerevisiae and related species, are very effective in fermenting glucose-based feedstocks into ethanol through anaerobic fermentation. However, these glucose-fermenting yeasts are unable to ferment xylose or L-arabinose, and are unable to grow solely on these pentose sugars. Although other yeast species, such as Pichia stipitis and Candida shehatae, can ferment xylose to ethanol, they are not as effective as Saccharomyces for fermentation of glucose and have a relatively low level of ethanol tolerance. Thus, the present range of available yeast are not entirely suitable for large scale industrial production of ethanol from biomass.
Most bacteria, including E. coli and Bacillus subtilis, utilize L-arabinose for aerobic growth, but they do not ferment L-arabinose to ethanol. These and other microorganisms, such as Zymomonas mobilis, have also been genetically modified to produce ethanol from hexose or pentose. This has been reported, for example, in Deanda, K., M. Zhang, C. Eddy, and S. Picataggio. 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl. Environ. Microbiol. 62:4465-4470; and Zhang, M., C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio. 1995 Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267:240-243. However, it remains the case that the low alcohol tolerance of these non-yeast microorganisms limits their utility in the ethanol industry.
Much effort has been made over the last decade or so, without truly overcoming the problem of developing new strains that ferment xylose to generate ethanol. Such efforts are reported, for example, in Kotter, P., R. Amore, C. P. Hollenberg, and M. Ciriacy. 1990. Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr. Genet. 18:493-500; and Wahlbom, C. F., and B. Hahn-Hagerdal. 2002 Recent studies have been conducted on yeast strains that potentially ferment arabinose. Sedlak, M., and N. W. Ho. 2001. Expression of E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae, Enzyme Microb. Technol. 28:16-24 discloses the expression of an E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae. Although this strain expresses araA, araB and araD proteins, it is incapable of producing ethanol.
U.S. patent application Ser. No. 10/983,951 by Boles and Becker discloses the creation of a yeast strain that may ferment L-arabinose. However, the overall yield is relatively low, at about 60% of theoretical value. The rate of arabinose transport into S. cerevisiae may be a limiting factor for complete utilization of the pentose substrate. Boles and Becker attempted to enhance arabinose uptake by overexpressing the GAL2-encoded galactose permease in S. cerevisiae. However, the rate of arabinose transport using galactose permease was still much lower when compared to that exhibited by non-conventional yeast such as Kluyveromyces marxianus. Another limitation that may have contributed to the low yield of ethanol in the modified strain of Becker and Boles is the poor activity of the L-arabinose isomerase encoded by the bacterial araA gene. Although Becker and Boles used an araA gene from B. subtilis instead of one from E. coli, the specific activity of the enzyme was still low. Other workers in the field have reported that low isomerase activity is a bottleneck in L-arabinose utilization by yeast.
There remains a need for new arabinose-fermenting strains that are capable of producing ethanol at high yield. There is further a need to identify novel arabinose transporters for introduction into Saccharomyces cerevisiae to boost the production of ethanol from arabinose.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.